Adenosine and guanosine are naturally-occurring and structurally similar purines. Moreover, naturally- occurring cyclic analogues of adenosine and guanosine are involved in similar physiological processes (e.g., both cAMP and cGMP mediate vasodilation). Strangely, at first blush it would seem that evolution broke the symmetry between adenosine and guanosine by bestowing cells with adenosine, but not guanosine, receptors. Thus the standard view is that adenosine serves as a physiological regulator of cellular function via activation of its own cell-surface receptors, while guanosine is merely an idle bystander. The purpose of this application is to challenge this received view by proposing that guanosine also plays an important role in cellular physiology, not via its own receptors but rather by engaging a powerful collaborative mechanism involving adenosine. In this regard, we hypothesize that extracellular guanosine is THE major physiological determinant of extracellular levels of adenosine because guanosine blocks the disposition of adenosine from the extracellular compartment allowing greater activation of adenosine cell-surface receptors due to increased extracellular adenosine levels. We refer to this guanosine-adenosine interaction as the GUANOSINE- ADENOSINE MECHANISM. Our pilot experiments strongly suggest that our hypothesis is correct. In preglomerular vascular smooth muscle cells (a model system for microvascular smooth muscle cells) we observe that for any given amount of adenosine added to cells, the extracellular concentrations of adenosine [as measured by high performance liquid chromatography-mass spectrometry (LC-MS/MS)] are more than 10- fold higher when guanosine is also added to the cells. We also find that cellular stress/injury increases extracellular levels of BOTH guanosine and adenosine. Moreover, we find that guanosine PROFOUNDLY augments the biological effects of adenosine in vivo! These findings suggest that extracellular guanosine, by blocking the disposition of extracellular adenosine, is the most important physiological determinant of extracellular levels of adenosine yet discovered. We propose to test the existence of, the mechanism of and the importance of the Guanosine-Adenosine Mechanism with the following six Specific Aims: Aim 1 - To determine the existence of the Guanosine-Adenosine Mechanism in vascular and renal cell types; Aim 2 - To determine whether the Guanosine-Adenosine Mechanism is mediated directly by guanosine or indirectly by conversion of guanosine to guanine; Aim 3 - To determine whether the Guanosine-Adenosine Mechanism involves inhibition by guanosine of enzymes involved in adenosine metabolism; Aim 4 - To determine whether the Guanosine-Adenosine Mechanism involves inhibition by guanosine of transporters involved in adenosine uptake; Aim 5 - To determine whether the Guanosine-Adenosine Mechanism importantly contributes to elevated extracellular levels of adenosine following cellular stress with energy depletion; and Aim 6 - To determine whether the Guanosine-Adenosine Mechanism amplifies the effects of adenosine in vivo. PUBLIC HEALTH RELEVANCE: Adenosine is a critical retaliatory metabolite. During cellular/tissue stress and injury, extracellular adenosine levels are elevated and activate cell surface receptors that mediate tissue/cellular protection (or in some cases participate in adverse responses). Our pilot experiments show that cellular stress/injury increases extracellular levels of guanosine as much as or more than extracellular levels of adenosine and that extracellular guanosine elevates the levels of extracellular adenosine by interfering with the disposition of adenosine from the extracellular compartment. Manipulation of this mechanism, which we call this the Guanosine-Adenosine Mechanism, can be exploited to treat diseases of the kidneys, heart, blood vessels, lungs, brain and immune system.